β-glucanase from a bacillus

ABSTRACT

A β-Glucanase enzyme capable of hydrolytically cleaving mixed glucans is presented. The β-Glucanase is sufficiently stable under alkaline conditions for use in industrial cleaning processes, especially in the brewing industry.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This application is filed under 35 U.S.C. 371 and based onPCT/EP98/04564, filed Jul. 21, 1998.

This invention relates to an enzyme capable of hydrolytically cleavingmixed glucans, which are linked alternately by 1,3- and 1,4-β-glucosidicbonds, into oligosaccharides and to the microorganism which forms thisenzyme.

Enzymes such as these belong to the class ofendo-1,3-1,4-β-D-glucan4-glucanohydrolases (EC 3.2.1.73; lichenases) orendo-1.3-β-D-glucosidases (EC 3.2.1.39; laminarinases). For the purposesof the present invention, an enzyme of this type is referred to hereinas β-glucanase or beta-glucanase.

2. Discussion of Related Art

Polymeric mixed glucans of the type mentioned above are present invarying amounts in virtually all cereal products. Enzymes capable ofcleaving them are required above all in the food, beverage and animalfeed industries, the textile industry and in the processing of starch(R. Borriss “μ-Glucan-spaltende Enzyme”, in H. Ruttloff: “IndustrielleEnzyme”, Chapter 11.5, Behr's Verlag, Hamburg (1994)). One of the mostimportant applications of β-glucanases is in the beverage and brewingindustries where enzymes such as these are used for degrading malt andbarley β-glucan. The enzymes used for this purpose normally emanate fromBacillus subtilis, as described for example in German patent DD 226 012A1, or from Bacillus amyloliquefaciens, although β-glucanases from othermicroorganisms, for example Achromobacter lunatus, Athrobacter luteus,Aspergillus aculeatus, Aspergillus niger, Disporotrichum dimorphosporum,Humicola insolens, Penicillium emersonli, Penicillium funiculosum orTrichoderma reesei, are also known. A commercial product intended foruse in the brewing industry is marketed, for example, under the name ofCereflo® (manufacturer: Novo Nordisk A/S).

Hitherto known β-glucanases have pH optima in the weakly acidic toneutral range, so that their use is confined to processes which arecarried out at those pH values. The problem addressed by the presentinvention was to extend the field of application of β-glucanases and todevelop a β-glucanase which would be sufficiently stable under alkalineconditions for use in industrial processes carried out under conditions.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts in one-letter code the amino acid sequence of theβ-glucanase according to the invention (SEQ ID-NO.:1) obtained fromBacillus alkalophilus DSM 9956.

FIG. 2 depicts glucanolytic activity as a function of pH for theβ-glucanase obtained from Bacillus alkalophilus DSM 9956 in Example 1below.

FIG. 3 depicts depicts glucanolytic activity as a function oftemperature for the β-glucanase obtained from Bacillus alkalophilus DSM9956 in Example 1 below.

DESCRIPTION OF THE INVENTION

The present invention relates to an enzyme obtainable from Bacillusalkalophilus DSM 9956 which has the glucanolytic activity mentioned atthe beginning, to the microorganism Bacillus alkalophilus DSM 9956 whichproduces a β-glucanase and to the gene encoding the ,β-glucanase fromBacillus alkalophilus DSM 9956 which was identified and sequenced (SEQID-NO.:2) in the course of the work culminating in the presentinvention. If desired, this gene may be cloned in known manner in otherbacteria and the β-glucanase may be expressed there. Accordingly, thepresent invention also relates to host organisms containing the saidgene obtainable by essentially microbiological processes. The amino acidsequence—derived from the sequence of the β-glucanase gene from Bacillusalkalophilus DSM 9956—of the β-glucanase according to the inventionobtainable from that microorganism (SEQ ID-NO.:1) is shown in theone-letter code in FIG. 1. The β-glucanase from Bacillus alkalophilusDSM 9956, including the signal peptide, which is split off by a signalpeptidase after transport through the cell wall of the microorganism andwhich, according to comparisons with data known from the literature[M.E. Louw, S. J. Reid, Watson, Appl. Microbiol. Biotech. 39 (1993),507-513], presumably comprises 31 amino acids, consists of 308 aminoacids. The corresponding microorganism is gram-positive, its cell formis rodlet-like (width ca. 0.7 μm to 0.9 μm, length ca. 2.5 μm to 4.0μm); it was deposited by applicants on 13.04.1995 in the DSM—DeutscheSammiung von Mikrooganismen und Zellkulturen GmbH, Mascheroder Weg 1b,38124 Braunschweig, and has been given the number DSM 9956.

A β-glucanase according to the invention preferably has a homology ofmore than 70%, more particularly 75% to 99%, to the β-glucanase fromBacillus alkalophilus DSM 9956. The same applies to the basic gene.

The enzyme according to the invention is preferably used in the foodindustry, more especially in the beverage and brewing industry, moreparticularly for removing glucan and/or lichenan in the cleaning ofmembranes and other equipment in those industries.

The present invention also relates to a process for removing glucanand/or lichenan in the cleaning of membranes and other equipment in thefood industry, more particularly the brewing industry, using aβ-glucanase according to the invention.

EXAMPLES Example 1

Chromosomal DNA from Bacillus alkalophilus C/M2-3 was partly digestedwith Sau3A and a fraction of 4 to 8 kb large fragments was isolated bygel electrophoresis. After ligation into the BamH1-site of the plasmidpMK4, an E. coli—Bacillus shuttle vector [M. A. Sullivan et al., Gene 29(1984), 21-26], it was transformed into competent E. coli DH5α cells.Recombinant clones with β-glucanase activity were identified by coloringwith Congo Red on LB plates containing 0.2% lichenin (pH 8.5).

The β-glucanase was purified from the cell supernatant of a clone in E.coli DH5α pmK4. After dialysis of the cell-free supernatant against 20mM sodium phosphate buffer (pH 7.5), the dialyzate was fixed toQ-Sepharose (Pharmacia) and eluted with a linear gradient of 0-1 M NaClin 25 mM sodium phosphate buffer (pH 7.5 or pH 9.0).

The detection and determination of the glucanolytic activity was basedon modifications of the process described by M. Lever in Anal. Biochem.47 (1972), 273-279 and Anal Biochem. 81 (1977), 21-27. A 0.5% by weightsolution of β-glucan (Sigma No. G6513) in 50 mM glycine buffer (pH 9.0)was used for this purpose. 250 μl of this solution are added to 250 μlof a solution containing the agent to be tested for glucanolyticactivity and incubated for 30 minutes at 40° C. 1.5 ml of a 1% by weightsolution of p-hydroxybenzoic acid hydrazide (PAHBAH) in 0.5 M NaOH,which contains 1 mM bismuth nitrate and 1 mM potassium sodium tartrate,are then added, after which the solution is heated for 10 minutes to 70°C. After cooling (2 minutes/0° C.), the absorption at 410 nm isdetermined against a blank value at room temperature (for example with aUvikon® 930 photometer) using a glucose calibration curve. The blankvalue is a solution which is prepared in the same way as the measuringsolution except that the glucan solution is added after the PAHBAHsolution. 1 U corresponds to the quantity of enzyme which produces 1μmole of glucose per minute under these conditions.

The specific activity of the enzyme thus obtained amounted to 4390 mUper mg protein whereas the activity of the starting solution was lowerby a factor of 152. The enzyme was colored (silver coloring) as ahomogeneous band in SDS polyacrylamide gel electrophoresis.

With the aid of marker proteins (cytochrome c, equine myoglobin,chymotrypsinogen, ovalalbumin, bovine serum albumin) as an internalstandard, the molecular weight of the β-glucanase from Bacillusalkalophilus DSM 9956 was estimated by SDS polyacrylamide gelelectrophoresis to be about 30,000.

In isoelectronic focusing (pH 3 to 9), the isoelectric point of theβ-glucanase was found by activity coloring to be at pH 5.2.

EXAMPLE 2

pH Profile

The determination of glucanolytic activity at various pH values wascarried but in a Davies universal buffer (21.01 g citric acid . H₂O,13.61 g KH₂PO₄, 19.07 g Na₂B₄O₇. 10 H₂O, 12.11 g tris and 7.46 g KCl in1 l dist. water; 50 ml of this stock solution are adjusted to therequired pH with 0.4 N NaOH and made up with dist. water to 200 ml) at40° C. after incubation for 30 minutes. As can clearly be seen from thepH profile shown in FIG. 2 (relative glucanolytic activity, rel. A.,plotted against the pH), the enzyme is at its most active between pH 6and pH 10.5. The optimum lies at pH 9.

EXAMPLE 3

Temperature Profile

The dependence on temperature of the glucanolytic activity of theβ-glucanase obtained from Bacillus alkalophilus DSM 9956 was measured inglycine/NaOH at pH 9 after incubation for 15 minutes. The pH value ofthe test solution was adapted because the buffer has a dependence ontemperature of ca. pH 0.033 per °C. The maximum of the glucanolyticactivity is at 60° C., as shown in FIG. 3 where the relativeglucanolytic activity (rel. A.) of the enzyme is plotted against thetemperature (T).

Statement Under 37 C.F.R. §§ 1.821(f) and (g)

The contents of the attached paper Sequence Listing and itscomputer-readable form are the same and add no new matter in thisapplication.

2 1 308 PRT Bacillus alkalophilus DSM 9956 1 Met Lys Arg Lys Thr Phe ValLeu Phe Ser Leu Phe Thr Leu Leu Ile 1 5 10 15 Gly Met Phe Ser Thr GlyPhe Ala Asn Thr Gly Val Val Gln Ala Glu 20 25 30 Asp Gly Arg Pro Met GlySer Thr Phe His Glu Thr Phe Asp Thr Phe 35 40 45 Asn Thr Asp Arg Trp SerThr Ala Gly Val Trp Thr Asn Gly Ala Met 50 55 60 Phe Asn Ala Thr Trp TyrPro Glu Gln Val Thr Ile Ser Asp Gly Lys 65 70 75 80 Met Lys Leu Gln IleAsp Lys Glu Asp Asp Glu Asp Ala Thr Pro Glu 85 90 95 Tyr Lys Ala Gly GluLeu Arg Thr Asn Gln Phe Tyr Gln Tyr Gly Leu 100 105 110 Phe Glu Val AsnMet Lys Pro Ala Lys Ser Thr Gly Thr Val Ser Ser 115 120 125 Leu Phe ThrTyr Thr Gly Pro Trp Asp Trp Asp Asn Asp Pro Trp Asp 130 135 140 Glu IleAsp Ile Glu Phe Leu Gly Lys Asp Thr Thr Arg Val Gln Phe 145 150 155 160Asn Tyr Phe Thr Asn Gly Val Gly Asn Asn Glu His Tyr His Glu Leu 165 170175 Gly Phe Asp Ala Ser Glu Ser Phe Asn Thr Tyr Ala Phe Glu Trp Arg 180185 190 Pro Glu Ser Ile Ser Trp Tyr Val Asn Gly Glu Leu Val Tyr Thr Ala195 200 205 Thr Glu Asn Ile Pro Gln Thr Pro Gln Lys Ile Met Met Asn LeuTrp 210 215 220 Pro Gly Ile Gly Val Asp Gly Trp Thr Gly Val Phe Asp GlyGlu Asp 225 230 235 240 Thr Pro Val Val Thr Glu Tyr Asp Trp Val Arg TyrThr Pro Leu Glu 245 250 255 Glu Leu Asp Asn Asn Gly Glu Gln Pro Lys ProVal Val Pro Gly Lys 260 265 270 Pro Glu Lys Pro Gly Lys Pro Gly Lys AsnGln Lys Asn Gln Glu Asn 275 280 285 Gln Glu Asn Gln Lys Asn Gln Glu AsnGln Lys Asn Gln Lys Ile Arg 290 295 300 Lys Thr Ser Ser 305 2 927 DNABacillus alkalophilus DSM 9956 2 atgaaaagga agacatttgt attattttctttatttacgt tgttaattgg tatgttctca 60 acagggtttg caaatacagg tgtggttcaggcagaagatg ggagaccaat ggggtcgacg 120 tttcatgaaa cgtttgatac ctttaatacggaccgctggt caacagctgg ggtatggaca 180 aatggagcaa tgtttaatgc gacatggtatccagaacagg tgaccatttc agatgggaaa 240 atgaagttgc aaattgacaa ggaagatgatgaagatgcaa ccccagaata taaggctggg 300 gaattaagaa cgaatcagtt ttatcaatacgggttgtttg aagtcaatat gaagccagcg 360 aaatcaacag gaaccgtctc ttcactctttacatatacgg gtccatggga ttgggataat 420 gatccttggg atgaaatcga tattgagttccttggaaagg atacaacaag agtccaattt 480 aactatttta ctaacggagt aggaaacaatgaacattacc acgaattagg gttcgatgca 540 tcagaatctt ttaatacgta tgcttttgaatggagaccag aatcaattag ttggtacgta 600 aacggagaat tagtatatac agcaacagaaaatatcccgc aaacaccaca aaaaattatg 660 atgaacttat ggcctggaat tggagtggatggatggacag gcgtttttga cggagaagac 720 actccagttg taacggagta tgattgggtaaggtacactc cactagagga attagataat 780 aacggagaac aaccgaaacc tgtagtgccaggaaaaccag aaaaaccagg aaaaccaggg 840 aaaaaccaga aaaaccagga aaaccaggaaaaccagaaaa accaggaaaa ccagaaaaac 900 caaaaaatca gaaaaaccag tagttga 927

What is claimed is:
 1. A biologically pure culture ofβ-Glucanase-producing microorganism Bacillus alkalophilus DSM
 9956. 2.An isolated polypeptide with β-glucanolytic activity having the aminoacid sequence SEQ ID NO:1.
 3. An isolated polynucleotide which encodesthe polypeptide of claim
 2. 4. An isolated polynucleotide comprising thesequence SEQ ID NO:2 or a polynucleotide with more than 70 percenthomology to SEQ ID NO:2, wherein said polynucleotide encodes apolypeptide with β-glucanolytic activity.
 5. The polynucleotide of claim4 which is 75 to 99 percent homologous to the sequence reproduced in SEQID-NO:2.
 6. A host microorganism transformed with the polynucleotide ofclaim
 3. 7. An isolated polypeptide with β-glucanolytic activity and ahomology of more than 70 percent to the polypeptide with the amino acidsequence SEQ ID NO:1.
 8. The polypeptide of claim 7 with a homology of75 to 99 percent to the polypeptide with the amino acid sequence SEQ IDNO:1.
 9. An isolated polynucleotide which encodes the polypeptide ofclaim
 7. 10. The polynucleotide of claim 9 with SEQ ID NO:2 or apolynucleotide with more than 70 percent homology to that sequence. 11.The polynucleotide of claim 10 with a homology of 75 to 99 percent toSEQ ID NO:2.
 12. A host microorganism transformed with thepolynucleotide of claim
 9. 13. A process for cleaning membranes andequipment in the food industry comprising contacting membranes andequipment with the polypeptide of claim
 2. 14. The process of claim 13wherein said membranes and equipment are used in the brewing industry.15. The process of claim 13 wherein said enzyme aids in the removal ofglucan and/or lichenan.
 16. A process for cleaning membranes andequipment in the food industry comprising contacting membranes andequipment with the polypeptide of claim
 7. 17. The process of claim 15wherein said membranes and equipment are used in the brewing industry.18. The process of claim 16 wherein said enzyme aids in the removal ofglucan and/or lichenan.